The present invention relates to a novel class of compounds with aspartyl protease inhibitory properties. This invention in particular relates to a class of amino acid derivatives with HIV aspartyl protease inhibitory properties that have been characterized by specific structural and physicochemical characteristics. In addition, this invention relates to different pharmaceutical compositions comprising these compounds. The compounds and the pharmaceutical compositions of this invention have been demonstrated to inhibit the activity of HIV aspartyl protease. Accordingly, this inhibitory property may be advantageously used to provide compounds with antiviral properties against HIV viruses, including the HIV-1 and HIV-2 viruses.
The HIV (human immunodeficiency virus) retrovirus is the causative agent for AIDS (acquired immunodeficiency syndrome). Thus the HIV-1 retrovirus primarily uses the CD4 receptor (a 58 kDa transmembrane protein) to gain entry into susceptible cells, through high-affinity interactions between the viral envelope glycoprotein (gp 120) and a specific region of the CD4 molecule found in CD4 (+) T-helper lymphocytes and other cells carrying the receptor (Lasky L. A. et al., Cell vol. 50, p. 975-985 (1987)). HIV infection is characterized by a period immediately following infection called xe2x80x9casymptomaticxe2x80x9d which is devoid of clinical manifestations in the patient. Progressive HIV-induced destruction of the immune system then leads to increased susceptibility to opportunistic infections, which eventually produces a syndrom called AIDS-related complex (ARC) characterized by symptoms such as persistent generalized lymphadenopathy, fever, weight loss, followed itself by full blown AIDS.
After entry of the retrovirus into a cell, viral RNA is converted into DNA, which is then integrated into the host cell DNA. The reverse transcriptase encoded by the viral genome catalyzes the first of these reactions (Haseltine W. A. FASEB J. Vol. 5 2349-2360 (1991)). At least three functions have been attributed to reverse transcriptase: RNA-dependent DNA polymerase activity which catalyzes the synthesis of the minus strand DNA from viral RNA, ribonuclease H (RNase H) activity which cleaves the RNA template from RNA-DNA hybrids, and DNA-dependent DNA polymerase activity which catalyzes the synthesis of a second DNA strand from the minus strand DNA template (Goff S. P. J. Acq. Imm. Defic. Syndr., vol. 3 p. 817-831 (1990)). The double stranded DNA produced by reverse transcriptase, now called provirus, is then able to be inserted into host genomic DNA.
At the end of reverse transcription, the viral genome now in the form of DNA is integrated into host genomic DNA and serves as a template for viral gene expression by the host transcription system, which leads eventually to the production of new viral particles (Sakai, H al., J. Virol. Vol. 67, p. 1169-1174 (1993)). The preintegration complex consists of integrase, reverse transcriptase, p17 and proviral DNA (Bukrinsky et al., Proc. Nat. Acad. Sci. USA vol. 89, p. 6580-6584 (1992)). The phosphorylated p17 protein plays a key role in targeting the preintegration complex into the nucleus of the host cell (Gallay et al., Cell, vol. 80, p. 379-388 (1995)), a necessary step for integration to take place.
The primary RNA transcripts made from the provirus are synthesized by the host cell RNA polymerase II whose activity is modulated by two virus-encoded proteins called Tat and Rev. The viral proteins are formed as polyproteins.
Post-translational modifications of viral polyproteins include processing and glycosylation of Env (envelope) proteins, and myristylation of the N-terminal residue of the p17 protein in the Gag and Gag-Pol polyproteins. The Gag and Gag-Pol precursors will give rise after cleavage to structural proteins and viral enzymes. The viral protease is the enzyme responsible for the cleavage of polyproteins Gag and Gag-Pol into mature proteins, a step essential for virus infectivity.
A number of synthetic antiviral agents have been designed to block various stages in the replication cycle of HIV. These agents include compounds which interfere with viral binding to CD4 T-lymphocytes (for example, soluble CD4), compounds which block viral reverse transcriptase (for example, didanosine and zidovudine (AZT)), budding of virion from the cell (interferon), or the viral protease (for example Ritonavir and Indinavir). Some of these agents proved ineffective in clinical tests. Others, targeting primarily early stages of viral replication, have no effect on the production of infectious virions in chronically infected cells. Furthermore, administration of many of these agents in effective therapeutic doses has led to cell-toxicity and unwanted side effects, such as anemia, neurotoxicity and bone marrow suppression.
Anti-protease compounds represent the most recent drugs developed to block HIV replication. These compounds inhibit the formation of infectious virions by interfering with the processing of viral polyprotein precursors. Thus, the antiviral potential of HIV protease inhibition has been demonstrated using peptidic inhibitors. Such peptidic compounds, however, are typically large and complex molecules that tend to exhibit poor bioavailability and are not generally consistent with oral administration. Accordingly, the need exists for compounds that can effectively inhibit the action of viral proteases, for use as agents for preventing and treating chronic and acute viral infections, such as HIV. The problem of viral resistance also underlines the need for new drugs to fight HIV infections.
It would be advantageous to have a class of derivatives that are aspartyl protease inhibitors, and particularly, HIV aspartyl protease inhibitors.
The present invention relates to a class amino acid derivatives as well as their pharmaceutically acceptable derivatives (e.g. salts).
Accordingly, the present invention in accordance with one aspect thereof provides a compound of formula I 
(as well as pharmaceutically acceptable derivatives thereof) and when the compound of formula I comprises an amino group pharmaceutically acceptable ammonium salts thereof,
wherein W is selected from the group consisting of xe2x80x94(CH2)nxe2x80x94, and xe2x80x94CH2xe2x80x94XXxe2x80x94CH2xe2x80x94CH2xe2x80x94
wherein n is 1, 2, 3 , 4 or 5,
wherein XX is selected from the group consisting of O, NR5, S, SO and SO2wherein Cx is selected from the group consisting of xe2x80x94COOM, xe2x80x94COOR5, xe2x80x94CH2OH, xe2x80x94CONR5R6, xe2x80x94CONHOH, 9-fluorenylmethoxycarbonyl-lysyl-NHxe2x80x94CO, benzyloxycarbonyl, and tetrazolyl, wherein M is an alkali metal (e.g. Na, K, Cs, etc.) or an alkaline earth metal, wherein R1 and R3, the same or different, are selected (i.e. independently) from the group consisting of H, tert-butoxycarbonyl, a straight or branched alkyl group of 1 to 6 carbon atoms, a cycloalkylalkyl group having 3 to 7 carbon atoms in the cycloalkyl part thereof and 1 to 3 carbon atoms in the alkyl part thereof (e.g. cyclopropylmethyl, cyclopentylmethyl, cyclohexylmethyl, cycloheptylmethyl, etc.) an arylalkyl group of formula (2) 
and a heterocycle-alkyl group of formula heterocycle-(CH2)mxe2x80x94wherein R2 and R4 the same or different are selected (i.e. independently) from the group consisting of H, CHOxe2x80x94, CF3xe2x80x94, CH3COxe2x80x94, benzoyl, 9-fluorenylmethoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, 4-OH-7-CF3-quinoline-3-COxe2x80x94, 3-indole-CH2CH2COxe2x80x94, 3-indole-CH2COxe2x80x94, 3-indole-COxe2x80x94, 2-indole-COxe2x80x94, C6H5OCH2COxe2x80x94, (C6H5)2COHCOxe2x80x94, C6H5SCH2COxe2x80x94, C6H5CH2CH2CSxe2x80x94, cholesteryl-OCOxe2x80x94, 2-quinoline-COxe2x80x94, xanthene-9-COxe2x80x94, 4-C6H5CH2CH2CONHC6H4SO2xe2x80x94, 2-NO2C6H4CHCHCOxe2x80x94, 3-C5H4NCHCHCOxe2x80x94, 3-C5H4NCH2CH2COxe2x80x94, fluorene-CH2COxe2x80x94, camphor-10-CH2xe2x80x94SO2xe2x80x94, (C6H5)2CHxe2x80x94COxe2x80x94, fluorene-COxe2x80x94, 1-naphthyl-SO2xe2x80x94, 2-naphthyl-SO2xe2x80x94, fluorenyl-SO2xe2x80x94, phenanthryl-SO2xe2x80x94, anthracenyl-SO2xe2x80x94, quinoline-SO2xe2x80x94, 4-CH3COONHC6H4xe2x80x94SO2xe2x80x94, C6H5CHCHxe2x80x94SO2xe2x80x94, 4-NO2C6H4xe2x80x94SO2xe2x80x94, an aryalkyl group of formula (2) as defined above, a sulfonyl group of formula (3) 
a heterocycle-alkylsulfonyl group of formula heterocycle-(CH2)mxe2x80x94SO2xe2x80x94 and
a carbonyl group of formula (4) 
wherein T is selected from the group consisting of xe2x80x94(CH2)mmxe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94 and xe2x80x94CH2xe2x80x94CHxe2x95x90CHxe2x80x94
wherein D is selected from the group consisting of O, NR7 and S,
wherein m is 1, 2, 3 or 4,
wherein mm is 0, 1, 2, 3 or 4
wherein X, Y and Z, the same or different, are selected (i.e. independently) from the group consisting of H, a straight or branched alkyl group of 1 to 6 carbon atoms, F, Cl, Br, I, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94NH2xe2x80x94NHR5, xe2x80x94NR5R6, xe2x80x94NHCOR5, xe2x80x94NHCOheterocycle, heterocycle being as defined above, xe2x80x94OR5, xe2x80x94SR5, xe2x80x94SOR5, xe2x80x94SO2R5, xe2x80x94COOR5, xe2x80x94CH2OH, xe2x80x94COR5, and xe2x80x94NHCOAryl, Aryl being an unsubstituted phenyl group or a phenyl group substituted by one or more members of the group consisting of a straight or branched alkyl group of 1 to 6 carbon atoms, F, Cl, Br, I, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94NH2xe2x80x94NHR5, xe2x80x94NR5R6, xe2x80x94NHCOR5, xe2x80x94OR5, xe2x80x94SR5, xe2x80x94SOR5, xe2x80x94SO2R5, xe2x80x94COOR5, xe2x80x94CH2OH, xe2x80x94COR5,
wherein R5 and R6, are independently selected from the group consisting of H, and a straight or branched alkyl group of 1 to 6 carbon atoms
wherein R7 is selected from the group consisting of HOxe2x80x94, CH3Oxe2x80x94, NCxe2x80x94, benzyloxy, and H2Nxe2x80x94 and
wherein heterocycle is selected from the group consisting of heterocyclic groups comprising 5 to 7 ring atoms, said ring atoms comprising carbon atoms and from one to four heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, said heterocyclic groups being monocycylic, bicycylic or monocycylic fused with one or two benzene rings.
The present invention in particular relates to a compound of formula I as defined herein pharmaceutically acceptable derivatives thereof and where applicable or appropriate pharmaceutically acceptable salts thereof, wherein W is xe2x80x94(CH2)nxe2x80x94, n is 3 or 4 and D is O. In accordance with the present invention R2 may in particular be a sulfonyl group of formula (3) as defined herein.
The present invention in particular provides a compound of formula Ia 
(as well as pharmaceutically acceptable derivatives thereof) and when the compound of formula Ia comprises an amino group pharmaceutically acceptable ammonium salts thereof, wherein W, Cx, R1, R2, R3 and R4 are as defined herein.
The present invention more particularly provides a compound of formula Ib 
(as well as pharmaceutically acceptable derivatives thereof) and when the compound of formula Ib comprises an amino group pharmaceutically acceptable ammonium salts thereof, wherein Cx, n, R1, R2, R3 and R4 are as defined herein.
The present invention particularly relates to a compound of formula I, Ia or Ib as defined herein (as well as pharmaceutically acceptable derivatives thereof) and where applicable or appropriate pharmaceutically acceptable ammonium salts thereof,
wherein Cx is selected from the group consisting of xe2x80x94COOM, xe2x80x94COOR5, xe2x80x94CH2OH, xe2x80x94CONHOH, and benzyloxycarbonyl, wherein M is an alkali metal (e.g. Na, K, Cs, etc.) and R5 is as defined herein,
wherein R1 and R3, the same or different, are selected (i.e. independently) from the group consisting of H, a straight or branched alkyl group of 1 to 6 carbon atoms, a cycloalkylalkyl group having 3 to 7 carbon atoms in the cycloalkyl part thereof and 1 to 3 carbon atoms in the alkyl part thereof and an arylalkyl group of formula (2) as defined herein wherein Z and Y are each H, m is 1 and X is H, Br or F
wherein R2 and R4 the same or different are selected (i.e. independently) from the group consisting of H, 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, 4-OH-7-CF3-quinoline-3-COxe2x80x94, 3-indole-CH2CH2COxe2x80x94, 3-indole-CH2COxe2x80x94, 3-indole-COxe2x80x94, 2-indole-COxe2x80x94, C6H5CHCHCOxe2x80x94, C6H5CH2CH2COxe2x80x94, C6H5CH2CH2CH2COxe2x80x94, C6H5CH2CHCHCOxe2x80x94, C6H5OCH2COxe2x80x94, (C6H5)2COHCOxe2x80x94, C6H5SCH2COxe2x80x94, C6H5CH2CH2CSxe2x80x94, 4-HOC6H4CH2CH2COxe2x80x94, cholesteryl-OCOxe2x80x94, 2-quinoline-COxe2x80x94, fluorene-COxe2x80x94, xanthene-9-COxe2x80x94, 4-C6H5CH2CH2CONHC6H4SO2xe2x80x94, 4-NO2C6H4CHCHCOxe2x80x94, 3-NO2C6H4CHCHCOxe2x80x94, 2-NO2C6H4CHCHCOxe2x80x94, 2,3-(CH3O)2C6H3CHCHCOxe2x80x94, 3,4-(CH3O)2C6H3CHCHCOxe2x80x94, 2,5-(CH3O)2C6H3CHCHCOxe2x80x94, 2,5-(CH3O)2C6H3CH2CH2COxe2x80x94, 3,5-(CH3O)2C6H3CH2CH2COxe2x80x94, 3,4-(CH3O)2C6H3CH2CH2COxe2x80x94, 2,4-(CH3O)2C6H3CHCHCOxe2x80x94, 2,4-(CH3O)2C6H3CH2CH2COxe2x80x94, 3,4-(CH3O)2C6H3CHCHCOxe2x80x94, 2,3-(CH3O)2C6H3CH2CH2COxe2x80x94, 4-CH3OC6H4CHCHCOxe2x80x94, 4-CH3OC6H4CH2CH2COxe2x80x94, 2-CH3OC6H4CHCHCOxe2x80x94, 3-CH3OC6H4CHCHCOxe2x80x94, 3-CH3OC6H4CH2CH2COxe2x80x94, 2-CH3OC6H4CH2COxe2x80x94, 4-CH3C6H4CHCHCOxe2x80x94, 4-HOC6H4CHCHCOxe2x80x94, 3-NH2C6H4CH2CH2COxe2x80x94, 3-C5H4NCHCHCOxe2x80x94, 3-C5H4NCH2CH2COxe2x80x94, fluorene-CH2COxe2x80x94, camphor-10-CH2xe2x80x94SO2xe2x80x94, (C6H5)2CHxe2x80x94COxe2x80x94, 1-naphthyl-SO2xe2x80x94, 2-naphthyl-SO2xe2x80x94, fluorenyl-SO2xe2x80x94, phenanthryl-SO2xe2x80x94, anthracenyl-SO2xe2x80x94, quinoline-SO2xe2x80x94, 4-CH3COONHC6H4xe2x80x94SO2xe2x80x94, C6H5CHCHxe2x80x94SO2xe2x80x94, 4-NO2C6H4xe2x80x94SO2xe2x80x94, and a sulfonyl group of formula (3) as defined herein wherein T is xe2x80x94(CH2)mmxe2x80x94 wherein mm is 0 and wherein X, Y and Z, are independently selected from the group consisting of H, a straight or branched alkyl group of 1 to 6 carbon atoms, F, Cl, Br, I, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94NH2, and xe2x80x94COR5 , wherein R5 is as defined herein
The present invention particularly provides a compound of formula I, Ia or lb as defined herein (as well as pharmaceutically acceptable derivatives thereof) and when applicable or appropriate pharmaceutically acceptable ammonium salts thereof, wherein R1 is selected from the group consisting of isobutyl, cyclopropylmethyl and benzyl, wherein R2 is a sulfonyl group of formula (3) as defined above, wherein R3 is H and wherein Cx is selected from the group consisting of COOM, and COOR5, M being an alkali metal (e.g. Na, K Cs, etc.) and R5 being as defined herein.
The present invention for example provides a compound of formula Ib as defined herein pharmaceutically acceptable derivatives thereof and where applicable or appropriate pharmaceutically acceptable salts thereof, wherein n is 4, wherein R1 is selected from the group consisting of isobutyl, cyclopropylmethyl and benzyl, wherein R2 is a sulfonyl group of formula (3) as defined herein, wherein T is xe2x80x94(CH2)mmxe2x80x94, wherein mm is 0, wherein X, Y and Z the same or different, are selected (i.e. independently) from the group consisting of H, a straight or branched alkyl group of 1 to 6 carbon atoms, Br, NO2, NH2, and OR5, wherein R3 is H, wherein wherein Cx is selected from the group consisting of COOM, and COOR5, wherein M is an alkali metal (e.g. Na, K, Cs, etc.), wherein R5 is as defined herein and wherein R4 is selected from the group consisting of 9-fluorenylmethoxycarbonyl, 2,3-(CH3O)2C6H3CH2CH2COxe2x80x94, 2,4-(CH3O)2C6H3CH2CH2COxe2x80x94, 3-indole-CH2CH2COxe2x80x94, C6H5CH2CH2COxe2x80x94, C6H5SCH2COxe2x80x94, C6H5OCH2COxe2x80x94, xanthene-9-COxe2x80x94, 4-CH3OC6H4CH2CH2COxe2x80x94, 3-CH3OC6H4CH2CH2COxe2x80x94, 2-CH3OC6H4CH2CH2COxe2x80x94, 3-NH2C6H4CH2CH2COxe2x80x94 and 
The compounds of the present invention have an affinity for aspartyl proteases, in particular, HIV aspartyl protease. Therefore, these compounds are useful as inhibitors of such proteases. These compounds can be used alone or in combination with other therapeutic or prophylactic agents, such as antivirals, antibiotics, immunomodulators or vaccines, for the treatment or prophylaxis of viral infection.
According to the present invention, the compounds of this invention are capable of inhibiting HIV viral replication in human CD4+ T-cells, by inhibiting the ability of HIV aspartyl proteases to catalyze the hydrolysis of peptide bonds. These novel compounds can thus serve to reduce the production of infectious virions from acutely and chronically infected cells, and can inhibit the initial or further infection of host cells. Accordingly, these compounds are useful as therapeutic and prophylactic agents to treat or prevent infection by HIV-1 and HIV-2, which may result in asymptomatic infection, AIDS-related complex (ARC), acquired immunodeficiency syndrome (AIDS), AIDS-related dementia, or similar diseases of the immune system, and related viruses such as HTLV-I and HTLV-II, and simian immunodeficiency virus.
As mentioned above heterocycle refers to a stable 5-7 membered monocycle or bicyclic heterocycle; it may be optionally benzofused or heterocyclofused. Each heterocycle consists of carbon atoms and from one to four heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. As used herein, the terms xe2x80x9cnitrogen and sulfur heteroatomsxe2x80x9d include any oxidized form of nitrogen and sulfur, and the quaternized form of any basic nitrogen. The heterocyclic ring may be attached by any heteroatom or carbon atom of the cycle, which results in the benzimidazolyl, imidazolyl, imidazolinyl, imidazolidinyl, quinolyl, isoquinolyl, indolyl, pyridyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazinyl, quinoxolyl, piperidinyl, morpholinyl, xcex2-carbolinyl, tetrazolyl, thiazolidinyl, benzofuranyl, thiamorpholinyl, benzoxazolyl, oxopiperidinyl, oxopyrroldinyl, oxoazepinyl, azepinyl, isoxazolyl, tetrahydropyranyl, tetrahydrofuranyl, thiadiazolyl, thiadiazinyl, benzodioxolyl, thiophenyl, tetrahydrothiophenyl, nicoticoyl, morpholinecarbodithioyl and sulfolanyl.
As mentioned above R2 and R4 are each independently (i.e. same or different) selected from the above mentioned class of substituents; the may in particular be 9-fluorenylmethoxycarbonyl (Fmoc), tert-butoxycarbonyl (t-Boc), benzyloxycarbonyl (Cbz), 2-chlorobenzyloxycarbonyl (2-ClCbz), substituted arylSO2, substituted arylalkylSO2, heteroarylSO2, acyl, substituted arylalkylacyl or heteroalkylacyl groups.
The configuration of the asymmetric centre can be D, L and DL, preferably the configuration corresponding to that found in L-lysine and L-ornithine.
In addition, this invention provides pharmaceutical compositions in which these novel compounds of formula I derived from L- amino acids are used to inhibit aspartyl proteases, including HIV aspartyl proteases, thus providing protection against HIV infection.
The terms xe2x80x9cHIV proteasexe2x80x9d and xe2x80x9cHIV aspartyl proteasexe2x80x9d are used interchangeably and refer to the aspartyl protease encoded by the human immunodeficiency virus type 1 or 2. In a preferred embodiment of this invention, these terms refer to the human immunodeficiency virus type 1 aspartyl protease.
The term xe2x80x9cpharmaceutically effective amountxe2x80x9d refers to an amount effective in treating HIV infection in a patient.
The term xe2x80x9cprophylactically effective amountxe2x80x9d refers to an amount effective in preventing HIV infection in a patient. As used herein, the term xe2x80x9cpatientxe2x80x9d refers to a mammal, including a human.
The term xe2x80x9cpharmaceutically acceptable carrier or adjuvantxe2x80x9d and xe2x80x9cphysiologically acceptable vehiclexe2x80x9d refer to a non-toxic carrier or adjuvant that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof.
As used herein, the compounds of this invention, including the compounds of formula I are defined to include pharmaceutically acceptable derivatives thereof. A xe2x80x9cpharmaceutically acceptable derivativexe2x80x9d means any pharmaceutically acceptable salt, ester, or salt of such ester, of a compound of this invention or any other compound which, upon administration to a recipient, is capable of providing (directly or indirectly) a compound of this invention or an antivirally active metabolite or residue thereof.
The compounds of this invention contain one or more asymmetric carbon atoms and thus may occur as racemates and racemic mixtures, single enantiomer, diastereomeric mixtures and individual diastereoisomers. All such isomeric forms of these compounds are expressly included in the present invention. Each stereogenic carbon may be of the R or S configuration.
Combinations of substituents and variables envisioned by this invention are only those that result in the formation of stable compounds. The term xe2x80x9cstablexe2x80x9d, as used herein, refers to compounds which possess stability sufficient to allow manufacture and administration to a mammal by methods known in the art. Typically, such compounds are stable at a temperature of 40xc2x0 C. or less, in the absence of moisture or other chemically reactive conditions, for at least a week.
The compounds of the present invention as mention above include salts. Salts of the compounds of this invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases (e.g. salts of acidic compounds of formula I with bases). Salts derived from appropriate inorganic and organic bases include for example, alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and Nxe2x80x94(C1-4 alkyl)4+ salts.
This invention also envisions ammonium salts (i.e. salts of amino groups) such as for example halide acid salts (e.g. hydrochloride, hydrobromide, hydroiodide salts). Thus the invention envisions the quaternization of any basic nitrogen containing groups (i.e. amino group(s)) of the compounds disclosed herein. The basic nitrogen can be quaternized with any agents known to those of ordinary skill in the art including, for example, lower alkyl halides, such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates including dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, and aralkyl halides including benzyl and phenethyl bromides. Water or oil-soluble or dispersible products may be obtained by such quaternization.
Other examples of acid salts include: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonace, cyclopentanepropionate, digluconate, dodecylhydrogensulfate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycollate, hemisulfate, heptanoate, hexanoate, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthylsulfonate, nicotinate, nitrate, oxalate, pamoate, pectinate, perchlorate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate, and undecanoate.
The compounds of this invention are readily prepared using conventional techniques from commercially available and cheap starting materials.
The compounds of this invention are among the most readily prepared HIV protease inhibitors known at this point. Previously described HIV protease inhibitors are resulting from long synthetic sequences and contain more than six chiral centers, numerous peptide bonds and require air-sensitive reagents such as organometallic complexes to achieve their successful preparations. The very easy synthesis of the products described in this invention represent a marked advantage, especially for the large scale preparation of these compounds.
In the following the preparation of compounds in accordance with the present convention will be described with reference to a number of process schemes wherein the various starting reactants as well as products thereof are designated by reference numbers e.g. in scheme 1 the starting ornithine or lysine is designated with the reference number 1.
In general, amino acid derivatives of formula I are readily obtained from commercially available sources. Following the indications summarized in Scheme 1, the Nxcfx89-benzyloxycarbonyl blocking group of Nxcex1-(9-fluorenylmethoxycarbonyl)xe2x80x94Nxcfx89-benzyloxycarbonyl ornithine or lysine 1 is removed by a treatment with TFA in CH2Cl2 according to the indications found in protective groups in Organic Synthesis, 3rd Edition, p. 520-521 (T. W. Greene and P. G. M. Wuts (John Wiley and Sons, Inc. 1999). The intermediate is obtained by the evaporation of the solvent and then reacted with a sulfonyl chloride or an acyl chloride derivative in the presence of a base such as 1M potassium carbonate, affording after normal work-up the desired product 2 in excellent yields. Another possible starting material could be Nxcex1-tert-butoxycarbonyl-Nxcfx89- benzyloxycarbonyl-L-ornithine or L-lysine 1a with the removal of the tert-butoxycarbonyl group being also achieved by a treatment with TFA in CH2Cl2. Products 2 with the Fmoc or the t-Boc groups were obtained in excellent yields. 
where 
Scheme 2, below, illustrates the preparation of Nxcex1-isobutyl-Nxcex1-(substituted benzenesulfonyl-Nxcex5-(9-fluorenylmethoxycarbonyl) derivatives 9 from readily available material Nxcex1-tert-butoxycarbonyl-Nxcex5-benzyloxycarbonyl-L-lysine 3 . The esterification with methyl iodide is achieved by treatment of the potassium salt in DMF with methyl iodide. Removal of the tert-butoxycarbonyl group from product 4 is done by treatment with TFA in methylene chloride. Reductive alkylation of the free amino group with isobutyraldehyde utilizing sodium cyanoborohydride provided the Nxcex1-isobutylamino acid derivative 6. Reaction with a substituted benzenesulfonyl chloride provides the product 7, the HCl scavenger being triethylamine or diisopropylethylamine. Hydrolysis of the methyl ester is accomplished with sodium hydroxide in methanol providing the acid 8 in good yield. It should be noted that extensive epimerisation takes place in this base catalysed hydrolytic reaction. The DL derivative 8 is then submitted to hydrogenolysis to remove the terminal blocking group and the free amino group can then be acylated with 9-fluorenylmethyl chloroformate or N-(9-fluorenylmethoxycarbonyloxy) succinimide to provide the desired product 9 in its racemized form. At that step, use of a substituted sulfonyl chloride provided the corresponding sulfonyl derivative and an acylation of the same amino group with an acyl chloride or an activated acid provided the acylated derivative of general structure 9. 
The problem of racemization was resolved by the use of a benzyl ester to block the carboxylic acid instead of a methyl ester. An additional advantage is the simultaneous removal of the two blocking groups (ester and carbamate) by hydrogenolysis, thus shortening the sequence by one step. The scheme 3, outlined below exemplifies this approach clearly. 
Scheme 4 demonstrates another improved approach to similar derivatives in a much shorter sequence and provide higher yields and avoid the use of protection-deprotection steps. The starting material for this sequence is a readily available commercial product, L-xcex1-amino-xcex5-caprolactam 14. Reductive alkylation utilizing the sodium cyanoborohydride conditions provided the alkylated derivative 15 in 95% yield as a crystalline solid that can then be subjected to reaction with a substituted sulfonyl chloride in presence of triethylamine in methylene chloride. Product 16 was obtained in 87% yield. Treatment with 12N HCl and acetic acid for 2 hours at reflux provided the lysine derivative 17 quantitatively and the terminal amino group was then acylated with an acyl chloride or an activated carboxylic acid to provide compound 18. Scheme 4a illustrates a particular example of the process of scheme 4. 
Scheme 5 summarizes the work done to obtain derivatives of structure I where n is 1. The starting material is L-serine 19a. Treatment with DEAD and triphenyl phosphine provided the xcex2-lactone 20 that was then treated with ammonia in ethanol. The Nxcex1-tert-butoxycarbonyl-Nxcex2-amino propionic acid derivative was then reacted as usual with a substituted benzenesulfonyl chloride, providing product 21. The removal of the blocking group and its replacement by another one (v.g. Fmoc) provided compound 22. Scheme 5a illustrates a particular example of the process of scheme 5. 
Scheme 6 below relates to an alternative process whereby compounds of formula I as defined herein may be obtained wherein W is xe2x80x94CH2xe2x80x94XXxe2x80x94CH2xe2x80x94CH2xe2x80x94, XX being as defined herein. Thus reductive alkylation of L-serine methyl ester 19b may give rise to compound 23 which may be treated with a substituted benzenesulfonyl chloride to give a compound 24. Further treatment of compound 24 with tosyl chloride in dichloromethane and triethylamine may give rise to a xcex1, xcex2-unsaturated ester 25. Michael addition of a substituted ethylenediamine and saponification may give rise to compound 26. The xcex1, xcex2-unsaturated ester 25 may be treated with a variety of reagents to provide compounds containing a heteroatom as shown in Table 2 for compound nos. 205, 206 and 207. The chiral derivatives may also be obtained via ring opening of a xcex2-lactone derived form 24 to give pure L isomers 26. 
Scheme 7 provides a summary of the approach of products of structure I where n is 2. Again the starting material is a simple product L-homoserine 27. The amino group is protected by the tert-butoxycarbonyl group and treatmemt with diazomethane in ether provided derivative 28. The next sequence is the transformation of the hydroxyl group to an amino group, which is easily achieved by treatment of 28 with 4-methylbenzenesulfonyl chloride in pyridine and methylene chloride followed by displacement of the tosyl group by azide in DMF. The product 29 is then reduced by hydrogen gas in presence of 10% Pd/C and the resulting amino group is reacted with a substituted benzenesulfonyl chloride, providing an excellent yield of derivative 30. Its conversion to another group on the alpha amino group is performed as previously described by the removal of the tert-butoxycarbonyl group with TFA in methylene chloride and then reaction with 9-fluorenylmethyl chloroformate or N-(9-fluorenylmethoxycarbonyloxy) succinimide, providing the final compound 31. 
As it can be appreciated by the skilled artisan, the above synthetic schemes are not intended to be a comprehensive list of all means by which the compounds described and claimed in this application may be synthesized. Further methods will be evident to those of ordinary skill in the art.
The compounds of this invention may be modified by appending appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.
As discussed above, the novel compounds of the present invention are excellent ligands for asp artyl proteases, particularly HIV-1 protease. Accordingly, these compounds are capable of targeting and inhibiting late stage events in the replication, i.e.; the processing of the viral polyproteins by HIV encoded protease. Compounds according to this invention advantageously inhibit the ability of the HIV-1 virus to infect immortalized human T cells over a period of days, as determined by an assay of extracellular p24 antigenxe2x80x94a specific marker of viral replication (see, Meek et al., Nature, 343, pp. 90-92 (1990)).
In addition to their use in the prophylaxis or treatment of HIV or HTLV infection, the compounds according to this invention may also be used as inhibitory or interruptive agents for other viruses which depend on aspartyl proteases, similar to HIV or HTLV aspartyl proteases, for obligatory events in their life cycle. Such compounds inhibit the proteolytic processing of viral polyprotein precursors by inhibiting aspartyl protease. Because aspartyl protease is essential for the production of mature virions, inhibition of that processing effectively blocks the spread of virus by inhibiting the production and reproduction of infectious virions, particularly from chronically infected cells. The compounds of this invention advantageously inhibit aspartyl proteases, thus blocking the ability of aspartyl proteases to catalyze the hydrolysis of peptide bonds.
The compounds of this invention may be employed in a conventional manner for the treatment or prevention of HIV, HTLV, and other viruses, which depend on aspartyl proteases for obligatory events in their life cycle. Such methods of treatment, their dosage levels and requirements may be selected by those of ordinary skill in the art from available methods and techniques. For example, a compound of this invention may be combined with a pharmaceutically acceptable adjuvant for administration to a virally infected patient in a pharmaceutically acceptable manner and in an amount effective to lessen the severity of the viral infection.
Alternatively, the compounds of this invention may be used in vaccines and methods for protecting individuals against viral infection over an extended period of time. The compounds may be employed in such vaccines either alone or together with other compounds of this invention in a manner consistent with the conventional utilization of protease inhibitors in vaccines. For example, a compound of this invention may be combined with pharmaceutically acceptable adjuvants conventionally employed in vaccines and administered in prophylactically effective amounts to protect individuals over an extended period of time against viral infections, such as HIV infection. As such, the novel protease inhibitors of this invention can be administered as agents for treating or preventing viral infections, including HIV infection, in a mammal.
The compounds of this invention may be administered to a healthy or HIV-infected patient either as a single agent or in combination with other antiviral agents which interfere with the replication cycle of HIV. By administering the compounds of this invention with other antiviral agents which target different events in the viral life cycle, the therapeutic effect of these compounds is potentiated. For instance, the co-administered antiviral agent can be one which targets early events in the life viral cycle, such as cell entry, reverse transcription and viral DNA integration into cellular DNA. Antiviral agents targeting such early life cycle events include didanosine (ddI), zalcitabine (ddC), stavudine (d4T), zidovudine (AZT), polysulfated polysaccharides, sT4 (soluble CD4)xe2x80x94which blocks attachment or adsorption of the virus to host cellsxe2x80x94and other compounds which block binding of virus to CD4 receptors on CD4 bearing T-lymphocytes and other CD4(+) cells, or inhibit fusion of the viral envelope with the cytoplasmic membrane. Other retroviral reverse transcriptase inhibitors, such as derivatives of AZT, may also be co-administered with the compounds of this invention to provide therapeutic treatment for substantially reducing or eliminating viral infectivity and the symptoms associated therewith. Examples of other antiviral agents include ganciclovir, dideoxycytidine, trisodium phosphonoformate, eflornithine, ribavirin, acyclovir, alpha interferon and trimenotrexate. Additionally, non-ribonucleoside inhibitors of reverse transcriptase, such as TIBO or nevirapine, may be used to potentiate the effect of the compounds of this invention, as may viral uncoating inhibitors, inhibitors of trans-activating proteins such as tat or rev, antisense molecules or inhibitors of the viral integrase. These compounds may also be co-administered with other inhibitors of HIV aspartyl protease.
Combination therapies according to this invention exert a synergistic effect in inhibiting HIV replication because each component agent of the combination acts on a different site of HIV replication. The use of such combinations also advantageously reduces the dosage of a given conventional anti-retroviral agent that would be required for a desired therapeutic or prophylactic effect as compared to when that agent is administered as a monotherapy. These combinations may reduce or eliminate the side effects of conventional single anti-retroviral agent therapies while not interfering with the anti-retroviral activity of those agents. These combinations reduce the potential of resistance to single agent therapies, while minimizing any associated toxicity. These combinations may also increase the efficacy of the conventional agent without increasing the associated toxicity. Preferred combination therapies include the administration of a compound of this invention with AZT, 3TC, ddI, ddC, d4T or other reverse transcriptase inhibitors.
Alternatively, the compounds of this invention may also be co-administered with other HIV protease inhibitors such as Ro 31-8959 (Saquinavir/Fortovase; Roche), L-735,524 (Indinavir; Merck), AG-1343 (Nelfinavir; Agouron), A-84538 (Ritonavir; Abbott) and VX-478 (Amprenavir; Glaxo) to increase the effect of therapy or prophylaxis against various viral mutants or members of other HIV quasi species.
We prefer administering the compounds of this invention as single agents or in combination with retroviral reverse transcriptase inhibitors, or other HIV aspartyl protease inhibitors. We believe that the co-administration of the compounds of this invention with retroviral reverse transcriptase inhibitors or HIV aspartyl protease inhibitors may exert a substantial synergistic effect, thereby preventing, substantially reducing, or completely eliminating viral infectivity and its associated symptoms.
The compounds of this invention can also be administered in combination with immunomodulators (e.g., bropirimine, anti-human alpha interferon antibody, IL-2, GM-CSF, methionine enkephalin, interferon alpha, diethyldithiocarbante, tumor necrosis factor, naltrexone and rEPO) antibiotics (e.g., pentamidine isethionate) or vaccines to prevent or combat infection and disease associated with HIV infection, such as AIDS and ARC.
When the compounds of this invention are administered in combination therapies with other agents, they may be administered sequentially or concurrently to the patient. Alternatively, pharmaceutical or prophylactic compositions according to this invention may be comprised of a combination of an aspartyl protease inhibitor of this invention and another therapeutic or prophylactic agent.
Although this invention focuses on the use of the compounds disclosed herein for preventing and treating HIV infection, the compounds of this invention can also be used as inhibitory agents for other viruses that depend on similar aspartyl proteases for obligatory events in their life cycle. These viruses include, but are not limited to other AIDS-like diseases caused by retroviruses, such as simian immunodeficiency viruses, HIV-2, HTLV-I and HTLV-II. In addition, the compounds of this invention may also be used to inhibit other aspartyl proteases and, in particular, other human aspartyl proteases including renin and aspartyl proteases that process endothelin precursors.
Pharmaceutical compositions of this invention comprise any of the compounds of the present invention, and pharmaceutically acceptable salts thereof, with any pharmaceutically acceptable carrier, adjuvant or vehicle. Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethyleneglycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
The pharmaceutical compositions of this invention may be administered orally, parenterally by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. We prefer oral administration or administration by injection. The pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically acceptable carriers, adjuvants or vehicles. The term xe2x80x9cparenteralxe2x80x9d as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are amino acid, water, Ringer""s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as Ph. Helv. or a similar alcohol.
The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspension and solutions. In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.
The pharmaceutical compositions of this invention may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.
Topical administration of the pharmaceutical compositions of this invention is especially useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxy-ethylene or polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable neat formulation. Topically-transdermal patches are also included in this invention.
The pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
Dosage levels of between about 0.01 and about 25 mg/kg body weight per day, preferably between about 0.5 and about 25 mg/kg body weight per day of the active ingredient compound are useful in the prevention and treatment of viral infection, including HIV infection. Typically, the pharmaceutical compositions of this invention will be administered from about 1 to about 5 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the patient treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Preferably, such preparations contain from about 20% to about 80% active compound.
Upon improvement of a patient""s condition, a maintenance dose of a compound, composition or combination of this invention may be administered if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment should cease. Patients may, however, require intermittent treatment on a long-term basis, upon any recurrence of disease symptoms.
As the skilled artisan will appreciate, lower or higher doses than those recited above may be required. Specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the infection, the patient""s disposition to the infection and the judgment of the treating physician.
The compounds of this invention are also useful as commercial reagents which effectively bind to aspartyl proteases, particularly HIV aspartyl protease. As commercial reagents, the compounds of this invention, and their derivatives, may be used to block proteolysis of a target peptide, such as an aspartyl protease, or may be derivatized to bind to a stable resin as a tethered substrate for affinity chromatography applications. These and other uses which characterize commercial aspartyl protease inhibitors will be evident to those of ordinary skill in the art.
An Enzymatic assay for determining the inhibition constant (Ki) of synthetic compounds targeting the HIV protease may be carried out as follows: This is a fluorometric assay based on the cleavage by protease of a substrate carrying a donor group (EDANS) and an acceptor group (DABCYL) on each side of the cleavage site, interacting together through fluorescence resonance energy transfer (FRET). Cleavage of the substrate by protease stops energy exchange between the two groups, resulting in a time-dependent increase in fluorescence intensity that is linearly related to the extent of substrate hydrolysis.
The enzymatic assay is done at 31xc2x0 C. in white 96-well fluorescence microplates, in a total volume of 200 xcexcL per well. The apparatus used for analysis is a FL600 fluorescence microplate reader (Biotek Instruments). The reaction is run first in the absence of protease inhibitors for 4 min, using 156 xcexcL of buffer at pH 4.7 (sodium acetate 100 mM, NaCl 1 M, EDTA 1 mM, DTT 1 mM, dimethylsulfoxide 10%, and BSA 1 mg/mL), 20 xcexcL of substrate H-2930 from Molecular Probes (final concentration 10 xcexcM) and 20 xcexcL of recombinant HIV-1 protease (final concentration 2.18 nM) purchased from Bachem Bioscience. Excitation of the fluorophore is done at 340 nm and emission at 485 nm is recorded continuously during the reaction, allowing determination of the enzyme""s initial velocity (v0). At the end of the 4 min incubation, the potential inhibitor at a defined concentration in a volume of 4 xcexcL is added to the reaction, and fluorescence readings are taken for another 4 min, allowing determination of enzyme velocity (vi) in the presence of the inhibitory compound. Several concentrations of the putative inhibitors are tested in the assay. After calculation of v0 and vi, the inhibition constant (Ki) of the compound is determined using the equation of Henderson:       Vo    Vi    =      1    +                  [        I        ]                    Ki        app            
Where   Ki  =            Ki      app                      1        +                  [          S          ]                    Km      
and [I]=inhibitor concentration, [S]=substrate concentration,
Km=Michaelis-Menten constant, Kiapp=apparent Ki
Note that the Michaelis-Menten constant of HIV protease is determined by running the assay without inhibitors, using several concentrations of substrate, and plotting the results as a Cornish-Bowden graph with the ratio substrate concentration/velocity as the ordinate and substrate concentration as the abscissa. Graphs are traced and the Ki determined using GraphPad Prism software v. 3.0.
The compounds listed in Tables 1 and 2 below were prepared by following Schemes 1, 2, 3, 4, 5, 6 or 7 above or using reaction conditions known to those skilled in the art. The activities of the compounds are also listed in the same table demonstrating their potential usefulness. In Table 1 are shown compounds of formula Ia, as defined above, wherein W is xe2x80x94(CH2)nxe2x80x94 and wherein n, Cx, R1, R2, R3, and R4, are set forth for each compound mentioned therein. In Table 2 are shown compounds of formula Ia, as defined above, wherein W is xe2x80x94CH2xe2x80x94XXxe2x80x94CH2CH2xe2x80x94 and wherein Cx, R1, R2, R3, and R4, are set forth for each compound mentioned therein.
In the description herein, the following abbreviations are used:
In order that this invention be more fully understood, the following examples are set forth relating to the preparation of example compounds in accordance with the present invention. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way. When an example relates to the preparation of a compound identified in Table 1 or 2 above, the compound number used in Table 1 or 2 will appear after the name of the compound prepared in accordance to the example, additionally with respect to the compound numbers used in the tables of examples 80, and 81 these numbers identify the compounds as the compounds corresponding to that respective number which appears in Table 1.
Analytical thin layer chromatography (TLC) was carried out with 0.25 mm silica gel E. Merck 60 F254 plates and eluted with the indicated solvent systems. Preparative chromatography was performed either by flash chromatography, using Silica Gel 60 (EM Science) with the indicated solvent systems and a positive nitrogen pressure to allow proper elution, or by preparative thin layer chromatography, again employing E. Merck 60 F254 plates of 0.5, 1.0, or 2.0 mm thickness. Detection of the compounds was carried out by exposing eluted plates, analytical or preparative, to UV light and treating analytical plates either with a 2% p-anisaldehyde solution in ethanol containing 1% acetic acid and 3% sulfuric acid or with a 0.3% ninhydrin solution in ethanol containing 3% acetic acid, followed by heating.
Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AMX-2 500 MHz equipped with a reversed QNP probe. Samples were dissolved in deuterochloroform (CDCl3), deuteroacetone (acetone-d6) or deuterated dimethylsulfoxide (DMSO-d6) for data acquisition using tetramethylsilane (TMS) as internal standard. Chemical shifts are expressed in parts per million (ppm), the coupling constants J are expressed in hertz (Hz) and multiplicities (denoted as s for singlet, d for doublet, dd for doublet of doublets, t for triplet, q for quartet, m for multiplet, and br s for broad singlet).
The following compounds were prepared either from a derivative of a L-amino acid or, when indicated, from a derivative of a D-amino acid using the procedures summarized in Schemes 1, 2, 3, 4, 4a, 5, 5a, 6 or 7.